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Requirement for Nuclear Factor-B Activation by a Distinct Subset of CD40-Mediated Effector Functions in B Lymphocytes¹

Yina Hsing^* and Gail A. Bishop^2,*,,,§

^* Immunology Graduate Program, and Departments of Microbiology and Internal Medicine, University of Iowa, Iowa City, IA 52242; and ^§ Veterans Affairs Medical Center, Iowa City, IA 52242

	Abstract

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CD40 stimulation, which is crucial for generating an effective T-dependent humoral response, leads to the activation of transcription factors NF-AT (nuclear factor of activated T cells), AP-1 (activator protein-1), and NF-B (nuclear factor-B). However, which CD40-mediated B cell functions actually require activation of specific transcription factors is unknown. We examined the causal relationship between NF-B activation and CD40 effector functions by evaluating CD40 functions in the presence of an inducible mutant inhibitory B (IB) superrepressor. IBAA inhibited nuclear translocation of multiple NF-B dimers without the complicating effect of depriving cells of NF-B during development. This approach complements studies that use mice genetically deficient in single or multiple NF-B subunits. Interestingly, only a subset of CD40 effector functions was found to require NF-B activation. Both CD40-induced Ab secretion and B7-1 up-regulation were completely abrogated by expression of IBAA. Surprisingly, up-regulation of Fas, CD23, and ICAM-1 was partially independent, and up-regulation of LFA-1 was completely independent, of CD40-induced NF-B activation. For the first time, it is clear that distinct transcription factors are required for the dynamic regulation of CD40 functions.

	Introduction

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CD40 ligation provides B cells with an important costimulatory signal that together with B cell receptor (BCR)³ engagement and cytokine signals leads to B cell activation 1, 2 . The importance of CD40-CD40 ligand (CD40L) interaction is made evident by patients with mutations in CD40L 3, 4, 5, 6, 7 . This defect greatly increases an individual’s susceptibility to bacterial infections due to an inability to induce an effective T cell-dependent humoral immune response 3, 4, 5, 6, 7, 8 . CD40 engagement is important for induction of isotype switching 1, 2 , differentiation to Ab-secreting plasma cells 9, 10 , up-regulation of surface molecules important for B cell interaction with Th cells 1, 2 , and generation of memory 11 . These functions are a result of a number of CD40 signal transduction pathways (reviewed in 2 . CD40 signaling results in the activation of transcription factors including NF-B 12, 13 , nuclear factor of activated T cells (NF-AT) 14 , and AP-1 (activator protein-1) 14, 15 . However, the importance of each of these transcription factors in inducing CD40 effector functions is not clear.

NF-B is a dimeric transcription factor that plays a central role in the regulation of immune functions 16, 17 . Cellular stimuli, such as TNF and IL-1 18 ; foreign pathogens, such as HIV 19 and EBV 20 ; and tumorogenic cells 21, 22, 23 can use NF-B to activate cellular proliferation, differentiation, and survival 18 . NF-B-dependent gene transcription quickly results upon activation of a cytoplasmic pool of NF-B proteins 24, 25 that have been inactivated by associated IB (inhibitory B) proteins 26, 27 . Receptor stimulation may result in phosphorylation of IB 28 on serine residues at positions 32 and 36 29, 30 . Thereafter, IB is ubiquitinated 31, 32 and then degraded by the proteasome 28 , releasing NF-B to translocate to the nucleus and control gene expression. Constitutively inhibiting IB degradation 29 or inducing IB protein production 33, 34 effectively prevents NF-B activation. NF-B-dependent functions such as protection from TNF-induced apoptosis in tumor cells can be abrogated by a nondegradable mutant IB with both N-terminal serines substituted with alanines 22, 23 . Inhibiting NF-B activation in host immune cells with glucocorticoid drugs helps to prevent graft rejection by inducing the production of IB in host immune cells 33, 34 .

NF-B gene-targeted knockout mice and promoter analyses are additional approaches that have helped greatly to elucidate functions requiring NF-B activation. Development and immune function were investigated in mice made genetically deficient in one or two of the five different NF-B subunits. These studies conclude that while RelB 35, 36, 37 and p52 38, 39 NF-B subunits play important roles in dendritic cell and metalophilic marginal zone macrophage development and function, p50 40, 41 , RelA (p65) 42, 43 , and cRel 44, 45 NF-B subunits are predominantly important in B and T cell function. RelA, p50, and c-Rel subunits differentially participate in B cell proliferation, Ab secretion, and isotype switching 17 .

The role of NF-B in controlling inducible gene expression has also been studied by analyzing the promoters of many immunologically important genes. Careful analysis of promoters containing putative NF-B sites has revealed the importance of NF-B in the expression of VCAM-1 46, 47 , ICAM-1 48 , and IL-8 49 , among others. Promoter analysis reveals the importance of numerous transcription factors in gene expression. However, these studies are limited by difficulties involved in efficiently transiently transfecting cell lines of interest with reporter constructs and the inability to analyze more than one promoter at once.

CD40 ligation leads to the degradation of multiple IB molecules, including IB, IBß, and IB. To determine which CD40 functions are dependent upon the activation of NF-B dimeric complexes associated with IB, we inducibly expressed an IB superrepressor (IBAA) in two different B cell lines. Inducible expression of IBAA avoids the complications associated with B cells developing in an abnormal environment and allows us to study multiple genes simultaneously. We found that NF-B activation is required for CD40-mediated Ab production. CD40 functions that are important for T cell/B cell interaction, such as the up-regulation of B7-1 50 and ICAM-1 51, 52 , also depend on NF-B activation. CD40-mediated up-regulation of Fas 53, 54 , which serves to down-regulate the immune response, is partially abrogated by NF-B inhibition. However, CD40-induced up-regulation of LFA-1 and the activation of c-Jun kinase are independent of NF-B activation.

	Materials and Methods

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Cell lines

Mouse B cell lines, CH12.LX 55 and M12.4.1 56 , were cultured in RPMI 1640 containing 10% FCS, 10 µM 2-ME, and antibiotics (BCM-10). CH12.LX and M12.4.1 IBAA transfectants were maintained in BCM-10 supplemented with 400 µg/ml geneticin (Life Technologies, Grand Island, NY). Spodoptera frugiperda (Sf9) cells were maintained in Sf-900 II medium (Life Technologies) with antibiotics. Generation and use of Sf9 cell lines were previously described 57, 58, 59 .

Construction of human CD40 and IBAA molecules

Flag-tagged IBAA with serine to alanine substitutions at positions 32 and 36 was constructed by PCR mutagenesis and cloned into a version of pOPRSVICAT (Stratagene, La Jolla, CA) modified in our laboratory. The chloramphenicol acetyltransferase gene was excised from pOPRSVICAT, and a multiple cloning site was inserted in its stead. This modified version is referred to as pOPRSVmcs1.

Generation of mouse B cell transfectants

M12.4.1 and CH12.LX stable transfectants expressing EF1 promoter-driven lac repressor (LacI) construct (modified from the p3'SS construct from Stratagene, La Jolla, CA by replacement of the promoter) were generated and selected in hygromycin, as previously described 60 . LacI expression was verified by Western blotting of cell lysates using a LacI-specific polyclonal antiserum (Stratagene; catalogue 217449). Subsequently, M12.4.1 and CH12.LX LacI-expressing transfectants were supertransfected with IBAA construct and selected in medium containing geneticin to generate IPTG-inducible IBAA transfectants.

Antibodies

Hybridomas producing UC8-169 (Armenian hamster IgG), YN1/1.74 (anti-mouse ICAM-1, rat IgG2a), and M17/4.4.11.9 (anti-mouse LFA-1, rat IgG2a) mAbs were obtained from American Type Culture Collection (Manassas, VA). Abs were purified from hybridoma supernatant by either saturated ammonium sulfate precipitation or affinity purification 61 . B3B4 (anti-mouse CD23, rat IgG) and EM95.3 hybridomas (anti-mouse IgE, rat IgG2a) were kind gifts from Dr. Thomas Waldschmidt (University of Iowa, Iowa City, IA). The 1C10 hybridoma (anti-mouse CD40, rat IgG2a) was a kind gift from Dr. Frances Lund (Trudeau Institute, Saranac Lake, NY). Fluorescein-conjugated anti-mouse B7-1 (hamster IgG) and anti-trinitrophenyl (hamster IgG) were purchased from PharMingen (San Diego, CA). M17/4.4.11.9, YN1/1.74, B3B4, and EM95.3 were directly conjugated to fluorescein, as described 61 . Anti-IB (catalogue sc-847), anti-IBß (sc-969), and anti-IB (sc-7155) rabbit polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-flag epitope tag mAb (M2) was purchased from Eastman Kodak (New Haven, CT). Anti-mouse IgG-HRP and anti-rabbit IgG-HRP Abs were purchased from Bio-Rad (Hercules, CA).

Western blotting analysis

Cells (5 x 10⁶) were grown in BCM-10 or induced with 200 µM IPTG for 24 h. Cells were lysed in 1% Nonidet P-40 lysis buffer (50 mM Tris, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 0.02% NaN₃, 50 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 50 µg/ml PMSF, and 400 µM EDTA) and incubated for 30 min at 4°C. Samples were centrifuged at 14,000 x g for 15 min at 4°C. Supernatants were quantitated in a protein assay and then stored at -20°C. Lysates (100 µg/lane) were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were probed with anti-IB, anti-IBß, anti-IB rabbit polyclonal Abs, or anti-flag mAb (Eastman Kodak), followed by HRP-labeled goat anti-rabbit IgG Ab or HRP-labeled goat anti-mouse IgG Ab. Protein bands were visualized with a chemoluminescent detection system (Pierce, Rockford, IL).

Nuclear extraction and electrophoretic mobility shift assay (EMSA)

Cells (10⁷) were stimulated at a concentration of 10⁶ cells/ml with 1 µg/ml anti-mouse CD40 or isotype control Ab. Nuclear extracts were prepared from stimulated cells, as previously described 62 . Extracts were recovered, quantitated against a BSA protein standard, and then stored with 5 µg/ml antipain, 30 µg/ml leupeptin, 50 µg/ml aprotinin, and 400 µM sodium vanadate at -70°C.

dsDNA probes were end labeled with [-³²P]ATP using T4 polynucleotide kinase. A total of 5 µg nuclear extract was incubated with 0.25–0.5 ng probe for 30 min. The NF-B probe was described previously 62 . Samples were separated on a 5% native polyacrylamide gel at a constant current of 20 mA. x-ray film was exposed to dried gels overnight at -70°C.

c-Jun kinase assay

M12.4.1-inducible IBAA transfectants were incubated in the presence or absence of IPTG (200 µM) for 24 h and then stimulated with 3 µg anti-mouse CD40, isotype control Ab, or 10 µg/ml PMA and 1 µM ionomycin (Sigma, St. Louis, MO) for 5 min at 37°C. Cells were lysed in 200 µl lysis buffer 1 (50 mM HEPES (pH 7.8), 0.3 M NaCl, 1.5 mM MgCl₂, 1.2 mM EDTA, 0.1% Triton X-100, 20 mM ß-glycerophosphate, 100 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM NaVO₄, 1 mM PMSF, 2 µM pepstatin, 2 µg/ml aprotinin, 1 µg/ml leupeptin), and c-Jun kinase activity was measured as previously described 63 . Reactions were separated by SDS-PAGE. Gels were stained with Coomassie blue and dried. Phosphorylated c-Jun was visualized by autoradiography, and cpm was quantitated with the Packard Instant Imager (Packard Instrument, Downers Grove, IL).

Up-regulation of surface molecules

M12.4.1-inducible IBAA transfectants were incubated in the presence or absence of IPTG (200 µM) for 24 h. Cells were stimulated in a 24-well plate (100,000 cells/well) with 2 µg anti-mouse CD40 or isotype control Ab for 72 h. Cells were washed and then incubated with PBS containing 0.5% FCS, 0.02% sodium azide, and 2.5 mM EDTA at 4°C for 15 min. Subsequently, cells were stained with Abs, and surface expression of B7-1, CD23, Fas, ICAM-1, and LFA-1 was determined by flow cytometry on a Becton Dickinson (Mountain View, CA) FACScan, as previously described 59 .

Ab secretion assay

Ab secretion by CH12.LX transfectants was determined as previously described 64 . Cells were preincubated in a 96-well plate (1.5 x 10³ cells/well) in the presence or absence of 200 µM IPTG for 24 h and then exposed to indicated stimuli for an additional 48 h. Sf9-CD40L cells were used at a ratio of 4 B cells:1 Sf9 cell, a previously determined optimal ratio 60 . The quantity of IgM-secreting cells/10⁶ viable cells recovered at the end of the culture period was determined in a direct hemolytic plaque assay 65 . CH12.LX cells have surface and secreted IgM specific for phosphatidylcholine 66 . Sheep erythrocytes used as a source of phosphatidylcholine Ag were purchased from Elmira Biologicals (Iowa City, IA), and were used in cultures at 0.1% final concentration. Mouse rTNF- was purchased from Endogen (Woburn, MA). LPS was purchased from Sigma.

RNase protection assay

M12.4.1 lacI parent cells and IBAA transfectants were incubated in the presence or absence of 200 µM IPTG for 24 h. Cells (2 x 10⁵) were stimulated for 40 h with isotype control Ab or anti-mouse CD40 (1C10). Cells were lysed with Trizol (Life Technologies) and RNA was isolated according to manufacturer’s instructions. RNA (10 µg) was incubated at 90°C briefly and then hybridized overnight at 56°C with ³²P-radiolabeled B7-1 and L32 riboprobes. Riboprobes were transcribed with T7 RNA polymerase from B7-1 2U pcDNA3 (constructed by Dan Harms, University of Iowa) and L32 pGEM4Z plasmids 67 . Hybridized RNA samples were treated with RNase T1 and RNase A at 30°C for 45 min. Samples were then extracted with phenol chloroform once, and chloroform twice. RNA was precipitated with 100% ethanol and washed with 70% ethanol. Samples were separated on a 6% polyacrylamide sequencing gel, and x-ray film was exposed to dried gels overnight at -70°C.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inducible IBAA effectively inhibits NF-B activation

CH12.LX and M12.4.1 cells expressing the lac repressor protein were transfected with an IBAA construct under the control of an RSV promoter that contains two lac repressor binding sites. IBAA expression was normally repressed in these cells, but was induced within 24–48 h upon incubation with the inducer, IPTG (Fig. 1A). Overexpression of IBAA decreased endogenous IB expression, but had no aberrant effects on basal expression or CD40-induced degradation of endogenous IBß or IB (Fig. 1B and data not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Induced IBAA inhibits NF-B activation in M12.4.1 and CH12.LX cell lines. A, M12.4.1 and CH12.LX cells transfected with lac repressor (LacI), with or without IPTG-inducible flag epitope-tagged IBAA, were incubated in the presence or absence of IPTG (200 µM) for 24 h. Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membrane was blotted with anti-flag mAb and HRP-conjugated anti-mouse IgG Ab. B, M12.LacI parent cells and M12 IBAA clones 1A2 and 2D5 were incubated in the presence or absence of IPTG (200 µM) for 24 h. Cells were stimulated with 5 µg isotype control Ab (EM95) or anti-mouse CD40 mAb (1C10) for 45 min. Expression of IB and IB was detected in cell lysates by Western blot analysis with anti-IB and anti-IB polyclonal rabbit Ab and HRP-conjugated anti-rabbit IgG Ab. C, CH12.LacI parent cells and CH12 IBAA clone 1A2 transfectants were incubated in the presence or absence of IPTG (200 µM) for 24 h. Cells (107) were stimulated with 1 µg/ml isotype control Ab (EM95) or anti-mouse CD40 mAb (1C10) for 20 min. Nuclear extracts were tested for NF-B activation by EMSA with a radiolabeled NF-B probe (P, probe only). Data are representative of three similar experiments.

c-Jun kinase activity is unaffected by IBAA expression

To determine the effect of IBAA on other CD40 signal transduction pathways, we tested the ability of CD40 to activate c-Jun kinase in the presence of the IBAA molecule (Fig. 2). Jun kinase is known to phosphorylate various members of the Jun gene family that together with Fos proteins form the AP-1 transcription factor. M12.LacI cells or cells expressing IPTG-inducible IBAA were stimulated with anti-CD40 or isotype control Abs. PMA and ionomycin stimulation served as a positive control for activation of c-Jun kinase. Expression of lac repressor alone did not have any adverse effects on Jun kinase activation. M12.IBAA cells activated Jun kinase similarly in the presence or absence of IBAA. Therefore, the CD40 molecule is able to initiate signals leading to Jun kinase activation in the absence of NF-B activation, showing that expression of IBAA is not generally inhibitory to cell activation or to all CD40 responses.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. CD40-mediated c-Jun kinase activation is unaffected by IBAA expression. M12.LacI parent cells and M12.IBAA clone 2D5 were rested at 37°C for 30 min before stimulation. Cells (5 x 106) were stimulated with 3 µg/ml isotype control Ab (EM95), anti-mouse CD40 mAb (1C10), or PMA (10 µg/ml) and ionomycin (1 µM) for 5 min. Cell lysates were prepared and assayed for kinase activity by phosphorylation of a glutathione-S-transferase/c-Jun substrate, as described in Materials and Methods. Data are representative of three comparable experiments with two independently isolated IBAA clones.

IBAA abrogates CD40-mediated Ab secretion

CD40 signaling leads to Ab secretion by CH12.LX cells and can synergize with BCR signaling to enhance Ab secretion 58 . Our previous studies have shown that CD40 structural mutants that fail to activate NF-B also fail to induce Ab secretion 59, 62 . To move beyond this correlation and determine whether NF-B activation is required for CD40-mediated Ig secretion, we enumerated Ab-secreting cells in the presence or absence of inducibly expressed IBAA. The parent cell line CH12.LacI secreted IgM Ab in response to CD40L stimulation in either the presence or absence of IPTG. In contrast, induced expression of IBAA in two individual IBAA CH12.LAC transfectant subclones abrogated CD40-mediated Ab secretion (Fig. 3A). LPS- and TNF--mediated Ab secretion were also abrogated, albeit not completely (Fig. 3, B and C). The synergistic effect of BCR plus CD40 engagement in cells induced to express IBAA did not rescue the signaling defect (Fig. 3B). CD40-mediated Ab secretion thus requires NF-B activation.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. IBAA expression abrogates CD40-induced IgM secretion. A, CH12.LacI parent cells and CH12.IBAA transfectants were incubated in the presence or absence of IPTG plus or minus stimuli for 72 h, and IgM-secreting cells (pfc) were enumberated as described in Materials and Methods. Data are representative of three similar experiments. B, CH12.LacI parent cells and CH12 IBAA clone 1A2 were induced as above. Cells were stimulated with 50 µg/ml LPS, Ag (0.1% SRBC), and/or CD40L-Sf9 cells, and IgM-secreting cells were determined as described in Materials and Methods. Data are representative of three similar experiments. C, CH12.IBAA were induced as above. Cells were stimulated with 50 µg/ml LPS, anti-CD40 mAb, and/or 20 pg/ml rTNF-, and IgM-secreting cells were determined as above.

IBAA inhibits CD40-mediated up-regulation of B7-1, Fas, and ICAM-1, but not LFA-1

We previously demonstrated that CD40 stimulation of M12.4.1 cells leads to the up-regulation of a number of B cell surface molecules important in B cell/T cell interaction, including B7-1, CD23, Fas, ICAM-1, and LFA-1 59 . Induced expression of IBAA in M12.4.1 transfectants abrogated CD40 or dibutyryl cAMP-mediated up-regulation of the costimulatory molecule B7-1, but did not affect BCR-mediated B7-1 up-regulation (Fig. 4 and data not shown). These cells have not lost their intrinsic ability to up-regulate B7-1, since CD40 stimulation of the same subclones in the absence of IPTG led to B7-1 up-regulation comparable with that seen in M12.LacI parent cells (Fig. 4A). Inhibiting NF-B activation by expressing IBAA should abrogate function at the transcriptional level. To ensure that abrogation of B7-1 surface expression was due to a reduction in B7-1 transcripts, we performed RNase protection assays for B7-1 transcripts (Fig. 5). IBAA expression inhibited the up-regulation of B7-1 transcripts when IBAA expression was induced. Uninduced transfectants and parent cells up-regulated B7-1 transcripts within 40 h of CD40 stimulation.

View larger version (44K): [in this window] [in a new window]   FIGURE 4. IBAA differentially abrogates CD40-mediated surface molecule up-regulation. M12.lacI parent cells and M12 IBAA clone 1A2-expressing cells were incubated in the presence or absence of IPTG (200 µM) for 24 h. Cells (105/well) were stimulated with 1 µg/ml isotype control Ab (EM95) or anti-CD40 mAb (1C10) for 72 h. Cells were stained for surface molecules and analyzed by flow cytometry, as described in Materials and Methods. A, Flow cytometry profiles of cells stained with anti-B7-1 Ab. B, Flow-cytometric analysis, as in A, was performed for the molecules shown. Mean cell fluorescence shift (MCFS) was calculated by first subtracting the background isotype control-stained samples from each sample staining. Then the basal mean fluorescence was subtracted from the CD40-induced mean fluorescence of each molecule. The MCFS of both M12 IBAA clones were averaged together. Results are representative of three similar experiments.

View larger version (77K): [in this window] [in a new window]   FIGURE 5. IBAA abrogates CD40-mediated B7-1 up-regulation by inhibiting B7-1 gene expression. M12.LacI parent cells and M12 IBAA clone 1A2 were incubated in the presence or absence of IPTG (200 µM) for 24 h. Cells were stimulated at 2 x 106 cells/well for 32 h with 1 µg/ml isotype control Ab (EM95) or anti-CD40 mAb (1C10) in 10 ml total volume. RNA was isolated and analyzed as described in Materials and Methods. The L32 (ribosomal RNA) probe served as a control. Data are representative of three similar experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inducible repression of NF-B activation in two mature B cell lines has allowed us to elucidate the causal relationship between activation of a transcription factor and mature B cell functions, including Ab secretion and expression of molecules involved in cell-cell interactions. We found that CD40-mediated B cell effector functions are differentially dependent upon NF-B activation. CD40-induced up-regulation of B7-1, the T cell costimulator, and Ab production was completely abrogated by IBAA, while other functions, including up-regulation of CD23, ICAM-1, and Fas, were only partially inhibited. In contrast, CD40-mediated LFA-1 up-regulation and c-Jun kinase activation were independent of NF-B activation.

Studies with mice deficient in one or two NF-B subunits have elucidated important roles of individual NF-B subunits in both development and immune function. However, interpretation is complicated by redundancy among the NF-B subunits and the analysis of mature functions in cells that have developed in an abnormal environment. Additionally, genetically targeting an individual NF-B subunit abolishes both homo- and heterodimers of that subunit. Different NF-B dimers may have opposing effects on transcription. For example, binding of p50 homodimer or p50-cRel heterodimer to the germline 1 Ig promoter has been shown to inhibit transcription, while binding of p50-RelA or p50-RelB heterodimers activates transcription 70 . Thus, genetically targeting a single NF-B subunit may knock out both transcriptionally active and suppressive dimers. Our experimental model takes an alternative and complementary approach that reveals the roles of multiple NF-B subunits inhibited by IB in mature B cell functions.

CD40 ligation leads to activation of NF-B subunits following the degradation of a number of IB molecules. Previously, we demonstrated that IB and IBß are degraded within 5 min of CD40 stimulation 62 . IB and IBß then reappear in the cytoplasm within 1–2 h 62 . In this study, we have determined that CD40 stimulation also leads to the degradation of IB with a delay in kinetics (Fig. 1). IB is degraded after 30 min of CD40 stimulation, and expression returns after 3 h of stimulation. Expression of IBAA differentially affects the ability of CD40 to signal through NF-B subunits that bind these IB molecules. Since the expression of IB is itself maintained and induced by NF-B subunits 71, 72 , it is not surprising that expression of IBAA leads to a decrease in endogenous IB protein levels. NF-B subunits remain permanently associated with IBAA because IBAA cannot be phosphorylated on the N-terminal serines or degraded 29 . Any NF-B subunits released from the endogenous IB will quickly associate with free IBAA. It has also been reported that IB can dissociate NF-B subunits from DNA 68 . Thus, NF-B subunits bound to IB are inhibited through a reduction in endogenous IB, association with a nondegradable IB, reassociation with IBAA if released from endogenous IB, and possibly dissociation of DNA-bound NF-B proteins.

IBAA expression had little to no effect on endogenous IBß or IB expression levels, suggesting that NF-B plays a lesser role in regulating IBß and IB gene expression than IB. However, since IB and IBß bind predominantly to the same set of NF-B subunits (p50-RelA and p50-cRel heterodimers) 68, 69 , IBAA expression most likely inhibits IBß-dependent NF-B activation as well. In contrast, IBAA may not inhibit IB-bound NF-B subunits since IB binds to a different subset of NF-B subunits, RelA homodimers, and RelA-cRel heterodimers 73, 74 . However, it is possible that overexpression of IBAA could lead to forced association with subunits released following IB degradation.

To ensure that the expression of IBAA did not adversely affect the global ability of CD40 molecules to signal, we assessed CD40-mediated c-Jun kinase activation and found it to be normal. In contrast, CD40-mediated Ig secretion and B7-1 up-regulation were completely abrogated by IBAA expression. Additionally, inhibition of B7-1 protein expression was at the level of B7-1 transcripts.

Promoter analysis of specific genes has helped to elucidate the role of transcription factors in mediating basal and induced expression or repression. Promoter and enhancer analysis of the B7-1 gene has uncovered a single NF-B binding site, which is found approximately 3 kb 5' of the translational start site 75 . This enhancer element is responsive to LPS and dibutyryl cAMP stimulation 75 . Our studies demonstrate the importance of this enhancer element in B7-1 up-regulation mediated by CD40 stimulation or dibutyryl cAMP (data not shown). In contrast, BCR-mediated B7-1 up-regulation was unaffected by the superrepressor, suggesting that for some stimuli, NF-B activation is not required. The B7-1 promoter itself contains a large number of sites for other inducible factors, including AP-1 and NF-IL-6 76 . The importance of these factors in CD40-induced B7-1 up-regulation is currently unknown. It is possible that CD40-mediated c-Jun kinase activation plays a contributory role in CD40-mediated gene regulation.

While IBAA completely abrogated CD40-mediated Ab secretion, it only partially inhibited TNF-- and LPS-mediated Ab secretion. Possibly, CD40-mediated differentiation is more dependent upon NF-B activation, while two other stimuli that activate NF-B, TNF-, and LPS induce other transcription factors that may induce differentiation. Alternatively, TNF- and LPS may activate greater amounts of or different NF-B subunits that cannot be effectively inhibited by IBAA.

CD40-mediated up-regulation of CD23, ICAM-1, and Fas was partially abrogated by IBAA expression. This implies that these genes are not completely dependent upon newly induced NF-B and can be partially induced by other transcription factors. Alternatively, like B7-1 up-regulation, NF-B activation is also absolutely required to induce transcription; however, other NF-B subunits not inhibited by IBAA partially compensate. Some CD40-mediated signals, in contrast, do not require NF-B activation. For example, the ability of CD40 to up-regulate LFA-1 expression was unaffected by IBAA expression, demonstrating that CD40-induced LFA-1 up-regulation requires activation of transcription factors other than NF-B.

These data illustrate the importance, but not sufficiency, of NF-B activation in CD40-mediated B cell effector functions. It is clear that some CD40 functions do not require NF-B activation. However, impairment of NF-B subunits associated with IB results in a loss of a number of important B cell functions, including Ab secretion, and up-regulation of molecules required for interaction with T cells. Since IBAA may not repress all dimeric forms of NF-B, the role of NF-B subunits associated with other inhibitors such as IB and the intricate relationships with other IB molecules, their associated subunits, and their gene targets are worthy of further investigation. CD40 also activates NF-AT and AP-1. The roles of these transcription factors in CD40 B cell effector functions also require investigation.

	Acknowledgments

 
We are grateful to Luis Ramirez for technical advice and Drs. Bruce Hostager and Gary Koretzky for valuable discussion.

	Footnotes

 
¹ This work was supported by grants to G.A.B. from the National Institutes of Health (AI28847 and CA66570), and from the Veterans Affairs (Merit Review 383). Core support was provided by National Institutes of Health Grant DK25295 to the University of Iowa Diabetes and Endocrinology Research Center.

² Address correspondence and reprint requests to Dr. Gail A. Bishop, Department of Microbiology, University of Iowa, 3-570 BSB, Iowa City, IA 52242. E-mail address:

³ Abbreviations used in this paper: BCR, B cell receptor; AP-1, activator protein-1; CD40L, CD40 ligand; EMSA, electrophoretic mobility shift assay; HRP, horseradish peroxidase; IB, inhibitory B; IPTG, isopropyl-B-D-thiogalactopyranoside; NF-AT, nuclear factor of activated T cells.

Received for publication September 23, 1998. Accepted for publication November 25, 1998.

	References

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